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Activation of human endothelial cytoplasts induces translation of pre‐synthesized JunB mRNA
Author(s) -
Jiang Huimiao,
Schmid Douglas I,
Weyrich Andrew S,
Zimmerman Guy A,
Kraiss Larry W
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a652-a
Subject(s) - junb , cytoplast , microbiology and biotechnology , messenger rna , biology , gene expression , cytoplasm , gene , biochemistry
Eukaryotic gene expression is regulated at transcriptional and translational checkpoints in distinct cellular compartments. Analysis of the translational contribution to gene expression is complicated by co‐existing transcriptional control. Hypothesis We hypothesized that endothelial cytoplasts, as enucleated cells, would be useful in translational control studies in vascular cells by eliminating the influence of transcription. Methods Enucleated cytoplasts were obtained by centrifuging HUVEC on coverslips cell‐side down in the presence of cytochalasin B. Following a recovery period, biological changes in response to thrombin, TRAP, or histamine were analyzed with immunocytochemistry and in situ hybridization. Results Cytoplasts expressed eukaryotic initiation factor‐4E and ribosomal protein S6 at levels similar to intact HUVEC. Thrombin or histamine induced actin rearrangement in cytoplasts, demonstrating competence for transmembrane signaling. Phospho‐p38 staining was increased by thrombin and TRAP in cytoplasts, suggesting a functional p38‐signaling pathway in cytoplasts. Abundant JunB mRNA was detected in cytoplasts whereas JunB protein was nearly undetectable. However, newly synthesized JunB protein was readily apparent in cytoplasts one hour after stimulation with thrombin, TRAP, or histamine. Conclusions (1) These studies demonstrate that translational regulation of JunB occurs via signal‐dependent mobilization of pre‐synthesized cytoplasmic JunB mRNA to the ribosome, and (2) cytoplasts are useful constructs for the isolated study of translational control mechanisms in endothelial cells. Supported by NIH HL‐75507.